1. Field of the Invention
This invention relates to and has among its objects the provision of novel blood coagulation components and novel methods of making them. Further objects of the invention will be evident from the following description.
2. Description of the Prior Art
There are estimated to be 100,000 cases of congenital hemophilia in the United States. Of these, approximately 20,000 are cases of hemophilia B, the blood of such patients being either totally devoid of plasma thromboplastin component or seriously deficient in plasma thrombopolastin component. The disease therefore exists in varying degrees of severity, requiring therapy anywhere from every week up to once or twice a year. The completely deficient cases require replacement therapy once every week; the partially deficient cases require therapy only when bleeding episodes occur, which may be as seldom as once a year. The bleeding episodes in congenital, partially-deficient cases are generally caused by a temporarily acquired susceptibility rather than by injury alone. Intravenous injection of a sufficiently large amount of fresh plasma, or an equivalent amount of fresh blood, temporarily corrects the defect of a deficient subject. The beneficial effect often lasts for two or three weeks, although the coagulation defect as measured by in-vitro tests on the patient's blood appears improved for only two or three days. Such therapy with fresh plasma or fresh blood is effective but it has several serious drawbacks: (1) it requires ready availability of a large amount of fresh plasma; (2) requires hospitalization for the administration of the plasma; (3) a great many of the patients become sensitized to repeated blood or plasma infusions and ultimately encounter fatal transfusion reactions; (4) at best plasma can only partially alleviate the deficiency; and (5) prolonged treatment or surgery is not possible because the large amounts of blood or plasma which are required will cause acute and fatal edema.
Several investigators noted that the mixing of blood of certain hemophilic patients would result in the mutual correction of the clotting defect of each blood. Interpretation of these findings was eventually made by Aggeler and co-workers [Proc. Soc. Exptl. Biol. Med. 79:692-696 (1952)] and S. G. White et al. [Blood 8:101-124 (1953)]. These workers, studying a male patient with a severe hemorrahagic diathesis associated with a prolonged clotting time which was clinically indistinguishable from classic hemophilia, postulated the existence of a new clotting factor. Aggelar et al., recognizing that the new factor was a precursor of thromboplastin, named it Plasma Thromboplastin Component (PTC). The work was confirmed by Biggs et al. [Brit. Med. J. 2:1378-1382 (1952)] in England, who gave it the name Christmas Factor, and by Soulier and Larrieu [New Eng. J. Med. 249:547-553 (1953)] in France, who called it Antihemophilic Factor B. This factor is now officially designated Factor IX.
In collaboration with Aggeler et al., the first fractionated PTC preparation was prepared [Revue d'Hematologie 9:447-453 (1954)]. The PTC was absorbed on barium sulfate from a solution of Cohn Fraction IV and eluted with 0.34 M sodium citrate. The yields are very small, and the post-infusion in-vivo activity was only about one-fourth that predicted from in-virto assays by a prothrombin consumption test.
Later, in collaboration with Aggeler et al., a process was developed by which PTC was adsorbed onto barium sulfate from EDTA anticoagulated plasma, eluted with a sodium chloride-sodium citrate buffer, and further purified by cold ethanol fractionation. This preparation was used clinically with excellent results [Aggeler, P. M. et al. Trans. 6th Congress, Internat. Soc. Hematol. Grune & Stratton, N.Y., pgs. 490-497 (1956)]. The process was later published in detail, and it was pointed out that the process was never commercialized because the Albumin and Plasma Protein Fraction obtained as by-products were contaminated with potentially hazardous levels of barium [Hink, J. H. and Johnson, F. F., The Hemophilias, CH. 18, page 156, edited by Brinkhous, Univ. of North Carolina Press (1964)]. Biggs et al. in England [Brit. J. Haematol. 7:349-364 (1961)] and Janiak and Soulier [Thromb. et Diath. Haemorr. 8:406-424 (1962)] in France later prepared Factor IX by a similar process, substituting tricalcium phosphate for the barium sulfate. However, spontaneous formation of thrombin always occurred and it was always necessary to add heparin, both during and after processing to neutralize the potentially dangerous thrombin.
Tullis et al. [New Eng. J. Med. 273:667-674 (1965)] have prepared and studied a somewhat similar plasma fraction, which they refer to as "prothrombin complex". In the Tullis process, the blood was collected through a calcium-removing ion exchange resin, and the resulting resin-plasma adsorbed onto DEAE cellulose. Elution was accomplished with a sodium phosphate-sodium chloride buffer stabilized with EDTA. Clinical studies on the "prothrombin complex" are described in the reference, but other data characterizing the complex are not available.
In U.S. Pat. No. 3,717,708 (hereinafter referred to as '708) there is described a lyophilized mixture of coagulation components of human origin useful in the treatment of patients, who bleed due to a congenital or acquired deficiency of one or more of the coagulation Factors II, VII, IX, X, prepared from human plasma collected in a citrate anticoagulant, free of heparin, free of thrombin, free of the activated form of Factor X, free of depressor activity, free of anti-complement activity, and comprising the coagulation components Factors II, VII, IX, X in essentially the same proportions as normal human plasma and of a potency equivalent to a specific activity of more than 0.5 clinical unit of each component per milligram of protein.
The product of '708 is produced by a process which comprises the steps of applying Cohn Supernatant I, Method 6, from unmodified citrated human plasma onto an ion exchange resin consisting essentially of cross-linked dextran chains with diethylaminoethyl groups attached by ether linkages to the glucose units of the polysaccharide chains, and adsorbing thereon said coagulation components of the plasma; selectively eluting the column with ammonium bicarbonate solution of a pH of from about 7.3 to about 8.2 and increasing molarity; separating the eluate fraction containing coagulation components; freezing the separated eluate fraction and removing the ammonium bicarbonate therefrom by lyophilizing the frozen fraction.
A method of producing a blood-coagulation-promoting preparation from human blood plasma is described in U.S. Pat. No. 4,160,025. The product was termed FEIBA for Factor VIII Inhibitor Bypassing Activity substance. In the production of FEIBA citrated human plasma is treated with a water-insoluble inorganic coagulation-physiologically-surface-active substance in the absence of free calcium ions to generate FEIB-activity. After separation from the above substance the supernatant is treated with an anion exchanger to adsorb FEIBA and coagulation Factors II, VII, IX, and X. The adsorbed materials are eluted from the exchanger with 3% sodium chloride (0.51 mole per liter) and 0.1% trisodium citrate dihydrate (0.0033 mole per liter). The eluate is dialyzed, frozen, and lyophilized.
A concentrate containing Factors II, IX, and X was prepared by Dike et al. (British Journal of Haematology, 1972, Vol. 22, pages 469-490). Blood plasma was diluted to one-third its volume with distilled water and adsorbed onto DEAE-cellulose in a chromatographic column. The column was washed with 0.02 M sodium citrate and then eluted with an aqueous solution of 0.1 M sodium chloride, 0.01 M-phosphate, and 0.01 M-citrate to give a Factor VII concentrate. Next, the column was eluted with 0.25 M sodium chloride, 0.01 M-phosphate, and 0.01 M-citrate to yield a concentrate containing Factors II, IX, and X.
Middleton et al. in Vox Sang., 1973, Vol. 24, pages 441-456, described a therapeutic concentrate of coagulation Factors II, IX, and X from citrated, Factor VIII-depleted plasma. After dilution of the plasma to one-third its volume with water for injection, the plasma was contacted with a DEAE-cellulose column to adsorb the coagulation Factors. Then, the cellulose was washed with an aqueous solution containing 0.03 M-phosphate and 0.03 M-citrate and eluted with an aqueous solution containing 0.03 M-phosphate, 0.03 M-citrate, and 0.2 M sodium chloride to give a concentrate of Factors II, IX, and X. This concentrate contained thrombin in amounts greater than the limits set by the U.S. National Institutes of Health. The authors note that the presence of phosphate is required in the eluting medium.
In Vox Sang., 1977, Vol. 33. pages 37-50, Suomela et al. described a method for large-scale preparation of a coagulation Factor IX concentrate. Coagulation Factors II, VII, IX and X from human plasma are adsorbed onto DEAE Sephadex. The gel was washed with an aqueous buffer solution containing 0.16 M sodium chloride and 0.04 M-phosphate and then eluted with a mixture of 0.5 M and 1.0 M phosphate buffer to give a Factor IX concentrate. The Factor IX:Factor VII ratio of this product was about 2:1 to 3:1 and the Factor IX:NAPTT ratio was about 3:1. The thrombin activity of the above concentrate was lower than 0.001 Units per milliliter.